Human breast milk miRNA, maternal probiotic

supplementation and atopic dermatitis Melanie Rae Simpson1*, Gaute Brede2, Jostein Johansen2, Roar Johnsen1, Ola Storrø1, Pål Sætrom2,3, Torbjørn Øien1

*Presenting author: melanie.simpson@ntnu.no

Background

Perinatal probiotic ingestion has been shown to prevent atopic dermatitis (AD) in

infancy.[1,2] The clinical trials underpinning this conclusion used a variety probiotics

strains and supplementation regimes, and the strongest evidence comes from studies

which include both pre- and post-natal supplementation. Results from our own study,

Probiotics in Prevention of Allergy among Children in Trondheim (ProPACT), suggest

that maternal supplementation alone is sufficient to prevent AD in offspring.[3] The

mechanisms behind this preventative effect are incompletely understood and may be

partially mediated by changes in breast milk composition. Micro-RNAs (miRNA) are

short non-coding RNA molecules which modify gene expression at the post-

transciptional level. They are abundant in mammalian milk and may influence the

developing gastrointestinal and immune systems of newborn infants.

Objectives

1. Describe the miRNA profile of human breast milk,

2. investigate the association between miRNAs and maternal probiotic supplementation, and

3. Determine if changes in breastmilk miRNAs are mediators of the beneficial effect of maternal

probiotic supplementation on the development of atopic dermatitis in offspring.

Methods

Conclusions

There appears to be a stable group of core breast milk miRNAs, which are at least partially conserved across a

number of mammalian species. Functional analysis of the potential gene targets of highly expressed breast milk

miRNAs revealed enrichment in a broad range of biological processes and molecular functions. Finally, individual

miRNAs in breast milk 3 months postpartum are unlikely to play a major role in the prevention of atopic dermatitis in

infancy by probiotics ingested in the perinatal period.

Results

Future plans

Current research in the ProPACT research group uses the various biological samples collected during the trial to

investigate the biological mechanisms by which maternal probiotic supplementation results in a reduced risk of

atopic dermatitis in offspring. We also plan to further investigate the general physiological role of breast milk

miRNAs and other non-coding RNAs through in vitro experiments and computational modelling. Particularly, with

respect to the other non-coding RNA (the tRNA and rRNA fragments) we will be conducting quality control

experiments using paired fresh and frozen samples to establish if these are observed because of storage.

Impact for the dairy industry

The ProPACT study provides scientific support for health

benefits of probiotic containing milk products. Indeed the

milk used in this study is a commercially available product

in Norway. More generally, the investigation of breast milk

constituents, such as miRNA, may prove to be of more

interest to the infant formula industry.

125 miRNAs

98.5% of reads

6568 targets

Top 20 miRNAs

76.0% of reads

3498 targets

Top 10 miRNAs

63.0% of reads

1759 targets

Top 5 miRNAs

53.8% 1371 predicted targets

Figure 3: Proportion of reads accounted for by the top

5, 10, 20 and 125 miRNAs and the number of

predicted unique target genes.

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Figure 1: Average percentage of small RNA sequences

aligned to different RNA species, with 95% CIs.

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Figure 2: Box plot of counts per million (CPM) mature

miRNA of the 20 most abundant miRNAs

Literature 1. Pelucchi C, Chatenoud L, Turati F, Galeone C, Moja L, Bach JF, et al. Probiotics

supplementation during pregnancy or infancy for the prevention of atopic

dermatitis: a meta-analysis. Epidemiology. 2012;23(3)402-414

2. Panduru M, Panduru NM, Salavastru CM, Tiplica GS. Probiotics and primary

prevention of atopic dermatitis: a meta-analysis of randomized controlled

studies. J Eur Acad Dermatol Venereol. 2015;29(2):232-42

3. Dotterud CK, Storrø O, Johnsen R, Øien T. Probiotics in pregnant women to

prevent allergic disease: a randomized, double-blind trial. Br J Dermatol. 2010;

163(3):616-23

1Department of Public Health and General Practice 2Department of Cancer Research and Molecular Medicine 3Department of Computer and Information Science

Participants

Fifty-four (54) mother-infant pairs were included in this analysis, 32 from the probiotics

group and 22 from the placebo group. Among the children, 11 and 18 had developed

atopic dermatitis by 2 years of age in the respective groups.

Small RNA profile of human breast milk

The RNA isolates obtained from the human milk samples contained between 12.4

and 247.5ng/µL of RNA, and were found to contain RNA fragments up to 1000nt.

Sequencing analysis of RNA molecules 11-28nt in length aligned primarily to rRNA,

tRNA and miRNA (Fig. 1).

miRNA profile

The miRNA profile revealed a relatively stable core group of highly expressed

miRNAs, including miR-148a-3p, miR-22-3p, miR-30d-5p, let-7b-5p and miR-200a-

3p (Fig. 2). These five miRNAs were responsible for over half of all miRNA found in

breast milk (Fig 3).

Functional predictions

The 20 most highly expressed miRNAs have 3498 predicted mRNA targets (Fig

3). Functional annotation clusters of these target genes included a) the positive

and negative regulation of metabolic processes involving nitrogen compounds,

RNA, DNA and macromolecules and the positive regulation of transcription and

gene expression, b) embryonic development, c) angiogenesis, d) catabolic

processes and e) cell migration and localisation. The first mentioned group of

clusters is large interwoven network of genes (Fig 4). There was also significant

overlap with genes that are upregulated in brain and epithelial tissues. Epithelial

cells in the gastrointestinal tract may be the first “recipients” of breast milk

miRNAs.

Differential expression associated with

maternal probiotic supplementation and

atopic dermatitis

Although several miRNAs were found to be

differentially expressed on comparison of the

probiotic vs placebo and AD vs non-AD groups,

none had an acceptable false discovery rate

(FDR) and their biological significance in the

development of AD is not immediately apparent

from their predicted functional consequences.

ProPACT Study

The Probiotics in the Prevention of Allergy among Children in Trondheim (ProPACT) trial is a

double blinded, placebo controlled, randomised trial investigating the effect of maternal probiotic

supplementation on the development of allergy related diseases in early childhood.[3]

2 yrs 1 yr Birth -4 wks 6 wks 3 mths

Intervention period

Biological samples collected from mother: • Stool samples (recuitment and 3mths)

• Vaginal swabs (recruitment and birth)

• PBMC and plasma (4 week before due date)

• Breast milk (10 days, 3 mths)

• Mouth swabs (10 days, 3mths)

Biological samples collected from child: • Cord blood (birth)

• Stool samples (10 days, 3mths, 1yr, 2yrs)

• PBMC and plasma (10 days, 3mths, 1yr, 2yrs)

• Mouth swabs (10 days, 3mths)

Lactobacillus rhamnosus GG (LGG; 5 x 1010 CFU)

Bifidobacterium animalis subsp. lactis Bb-12 (Bb-12; 5 x 1010 CFU)

L. acidophilus La-5 (La-5; 5 x 109 CFU).

Probiotic milk (n=211)

Placebo milk (n=204) Cultured milk, sterilised after culture process

PBMC: peripheral blood mononuclear cell

n=287 n=415

UCAGUGCACUACAGAACUUUGU

AAGCUGCCAGUUGAAGAACUGU

hsa-miR-148a-3p

hsa-miR-22-3p

Target genes and

their functions

Fifty-four breast milk samples from 3 months postpartum collected

during the ProPACT trial were used. Samples had been stored at -

80ºC for 7 to 9 years at time of analysis.

Extracellular vesicle and RNA isolation: breast milk samples

were enriched for extracellular vesicles using ExoQuick™, and total

RNA was isolated using Quigen miRNeasy kit.

Sequencing and bioinformatics pipeline: Small RNA sequencing

was conducted using 50-bp single-end reads on Illumina’s HiSeq

2000 sequencing system. Raw reads were processed, filtered and

mapped to the human genome and to miRNAs in miRBase version

20.0. Differential expression of miRNAs was assessed for the

probiotic vs placebo and the AD vs non-AD groups using the voom

and limma R-packages.

Functional predictions: Target genes were predicted for the 20

most highly expressed miRNAs and for the differentially expressed

miRNA lists using TargetScan (v7.0). Insight into the potential

functional consequences of these miRNA was gained by uploaded

gene target lists to the Database for Annotation, Visualization and

Integrated Discovery (DAVID) v6.7.

n=54

Trial registration and ethics approval: The clinical trial and this sequencing study were approved by the Regional

Committee for Medical Research Ethics for Central Norway (Ref. 097-03) and written consent was obtained from the

participating families. The original trial protocol is registered in ClinicalTrials.gov (identifier NCT00159523).

Figure 4: Enrichment map of the 50 highest ranked GO terms. The large network to the

left represents an interwoven network of terms associated with transcription, gene

expression, metabolic and biosynthetic processes. The red nodes represent negative

regulation of these processes, the green nodes positive regulation and the orange

nodes general regulation. Node size inversely weighted according to FDR. Edge colour

and width is weighted by similarity coefficient with wider and darker edges representing

a high coefficient.

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